Survival according to the site of metastasis in triple-negative breast cancer patients: The Peruvian experience

Background Evidence regarding differences in survival associated with the site of metastasis in triple-negative breast cancer (TNBC) remains limited. Our aim was to analyze the overall survival (OS), distant relapse free survival (DRFS), and survival since the diagnosis of the relapse (MS), according to the side of metastasis. Methods This was a retrospective study of TNBC patients with distant metastases at the Instituto Nacional de Enfermedades Neoplasicas (Lima, Peru) from 2000 to 2014. Prognostic factors were determined by multivariate Cox regression analysis. Results In total, 309 patients were included. Regarding the type of metastasis, visceral metastasis accounted for 41% and the lung was the most frequent first site of metastasis (33.3%). With a median follow-up of 10.2 years, the 5-year DRFS and OS were 10% and 26%, respectively. N staging (N2-N3 vs. N0, HR = 1.49, 95%CI: 1.04–2.14), metastasis in visceral sites (vs. bone; HR = 1.55, 95%CI: 0.94–2.56), the central nervous system (vs. bone; HR = 1.88, 95% CI: 1.10–3.22), and multiple sites (vs. bone; HR = 2.55, 95%CI:1.53–4.25) were prognostic factors of OS whereas multiple metastasis (HR = 2.30, 95% CI: 1.42–3.72) was a predictor of MS. In terms of DRFS, there were no differences according to metastasis type or solid organ. Conclusion TNBC patients with multiple metastasis and CNS metastasis have an increased risk of death compared to those with bone metastasis in terms of OS and MS.


Introduction
Among the phenotypes of breast cancer, triple-negative breast cancer (TNBC) is characterized by the lack of expression of estrogen, progesterone, and human epidermal growth factor 2 (HER2) receptors [1].TNBC is frequent in African American and Hispanic women [2], and accounts for 15-20% of all breast cancers [3].
This phenotype is well known for its aggressiveness, high heterogeneity, high percentage of metastasis to solid organs, and high relapse rates compared to other phenotypes.For instance, one quarter of patients with mild local disease present relapse with distant metastasis within the first three years after diagnosis [4].Due to these characteristics in addition to the lack of targeted therapy, TNBC has a poor prognosis with a worse overall survival and breast cancer cause specific-survival [5].
It is estimated that 20-30% of early cases of breast cancer will develop metastasis and the site of distant recurrence is associated with the breast cancer subtype [6].TNBC has a higher rate of metastasis in lung, bone and brain, as well as multiple metastasis [7,8].Although the literature has shown that TNBC generally has a lower OS and relapse-free survival (RFS) than other phenotypes, data on survival according to the site of distant metastasis is still limited.
Hence, our aim was to analyze the OS and distant relapse free survival (DRFS) according to the site of metastasis in women with TNBC.Moreover, as a secondary objective, we analyzed the survival since the diagnosis of the relapse (MS) in TNBC patients.

Study design and patient population
This was retrospective cohort study.We included and reviewed medical records from TNBC patients diagnosed and treated at the "Instituto Nacional de Enfermedades Neoplasicas" (Lima, Peru) from 2000 to 2014.The follow-up was until February 2020.The data were collected from December 2017 to March 2020.Inclusion criteria were as follows: age 18 years and older, breast cancer patients with confirmed a triple negative phenotype, stages I-III at diagnosis, and patients with distant relapse.The study included all the patients who met the inclusion and exclusion criteria.Cases lost to follow-up were excluded.
The type of metastasis was classified as visceral, central nervous system (CNS), bone, and multiple sites.Visceral metastasis was defined as the spread of cancer cells to adjacent or distant organ sites inside the thorax or abdominal cavity; whereas CNS metastasis included dura, brain parenchyma, and leptomeninges; while, bone metastasis referred to disease extension to bone.Multiple metastasis involved disease extension to multiple sites, such as bone and one solid organ or multiple solid organs.Later metastases were not considered in the initial classification.

Follow-up
OS was considered as from diagnosis of the primary tumor until death or the end of the study.DRFS was considered as from the date of the beginning of any type of treatment until the diagnosis of the first distant metastasis, death, or the end of the study.MS was considered from the diagnosis of distant metastasis to the death or the end of study.
Distant relapse of breast cancer was identified by computed tomography, magnetic resonance imaging, bone scintigraphy, or positron emission tomography.

Statistical analysis
For quantitative variables we used means and standard deviation (SD) or median with the interquartile range according to the distribution of data, while frequencies and percentages were estimated for qualitative variables.For univariate analysis, the Student's t or Mann-Whitney U test (according to the distribution) was used for quantitative variables, while the chisquare test was used for qualitative variables.Survival rates were calculated using the Kaplan-Meier method, while differences between types of metastases were assessed by the Log rank test.Missing data were reported but were not included in the statistical analysis.Prognostic factors and their hazard ratios (HR) were estimated with multivariate Cox regression analysis.To address any potential sources of bias, we included all the population available in our analyses.All the analysis had a 95% of confidence interval (95% CI), and a p value <0.05 was considered statistically significant.All the analyses were performed with the Statistical Package for the Social Sciences (SPSS) v. 24
Compared to visceral, CNS, or multiple metastasis; bone metastasis was more frequent in patients with a family history of breast or ovarian cancer (P = 0.02).N staging significantly differed among the four groups, with patients with multiple metastasis sites presenting similar lymph node involvement to that of patients with visceral and CNS metastasis, while patients with bone metastasis had a higher number of affected lymph nodes (P = 0.02).Finally, death was more frequent in the group with multiple metastasis sites (97.4%) compared to the groups of CNS (88.5%),visceral (85.6), and bone metastasis (81.6%) (P<0.01)(Table 3).
The study population was followed for a median of 10.2 years.The DRFS at 3 and 5 years was 30% and 10%, respectively (Fig 1 ), while the OS was 44% and 26%, respectively (Fig 1).
There were no differences when DRFS was assessed according to the type of metastasis (p = 0.08).Similarly, there were no differences when comparing the survival of patients with metastasis with lung, brain, or liver (p = 0.17) (Figs 2 and 3).However, the OS and MS rates were lower in patients with multiple sites of metastasis, followed by visceral, CNS, and bone metastasis at 3 years (34%, 44%, 62%, and 47%) and at 6 months (33%, 48%, 45%, and 61%), respectively (Fig 4).

Discussion
We evaluated the association between the site of metastasis site and clinical outcomes in patients with TNBC.The results showed that N staging, CNS metastasis to and multiple With regards to the demographic characteristics of TNBC patients, our results are consistent with previous literature.The mean age of our patients was 48 years old, being similar to previous studies describing the diagnosis of TNBC in patients no older than 50 years old.Indeed, TNBC is more frequent in younger women compared to other phenotypes of breast cancer [10,11].Hence, TNBC is mainly presented in pre-menopausal women, although this factor has not been associated with an impact on survival outcomes [12,13].
The majority of our patients were diagnosed in stage III (78.7%).TNBC patients are less likely to be diagnosed in a screening mammography, leading to diagnosis being made at a more advance stage [11,14].Moreover, In Latin America most cases are diagnosed in advanced stages due to the serious problem of limited access to breast cancer screenings in these countries [15,16] Asad et al. found that the stage at diagnosis was the most significant factor for the rapid development of relapse of TNBC; with 55% of patients with stage III TNBC at diagnosis developing relapse, demonstrating a 15-fold higher probability of relapse compared to women with stage I [17].The majority of our patients (78.1%) have a histologic grade III.In fact, almost all TNBC tumours are grade III invasive ductal adenocarcinomas [18,19].Cheung et al. reported a correlation between histological grade and the expression of the snail2 protein, which is linked to transformation of tumor cells to the mesenchymal phenotype, thereby enabling metastasis and suggesting an association between histological grade and TNBC metastasis [20].In spite of the fact that all the patients included in this study received either NAC, ACT, and/or radiotherapy, all of them developed relapse.In accordance with a prior cohort study about recurrence of TNBC; results showed that despite the majority of patients after undergoing adjuvant therapy, a dramatically high rate of recurrence was still observed [21].A possible explanation of this outcome may be found in the study by Kim et al., which showed that breast cancer cells can develop resistance or even have pre-existing resistance to chemotherapy [22].However, a meta-analysis by Yang et al. found that adjuvant chemotherapy reduced the rate of recurrence for T1a/bN0 TNBC [23].Albeit promising, these results cannot be extrapolated to our investigation as the patients in the aforementioned study were all T1 whereas more than the 95% of our patients were T2 and above.Although the current NCCN guidelines are well established for each tumour size and lymph node invasion [24], information on the efficacy of chemotherapy and reduction of recurrence in advanced TNBC stages is still limited, and therefore additional studies are needed to elucidate the true role of the chemotherapy in TNBC relapse.
Predominant distant metastasis is a documented finding in TNBC, Dent et al. found that women with TNBC had an increased likelihood of distant recurrence within five years after diagnosis [25].In fact, research suggests that the high risk and predominance of distant recurrence is in part caused by the excess of visceral and CNS metastases, but diminished bone recurrence seen in patients in the first five years after diagnosis [26,27].Some explanations for the tendency of TNBC to present visceral and CNS metastasis include the association between metastasis to certain organs and the expression of specific genes by the tumour [28].Hicks et al. described that breast cancer patients that developed brain metastasis were likely to express cytokeratin 5 and 6 markers [29].The expression of cytokeratin 5 is linked to the phenotype of cells undergoing the epithelial-mesenchymal transition, which is used by the tumour to gain migratory and invasive properties, thereby causing metastasis [30][31][32].We found that lung metastasis was the most common site of metastasis similar to what other authors described, and was also the most common first site of invasion, followed by multiple metastasis and brain [33][34][35][36][37][38].However, it has been proposed that the genes present in TNBC comprise the signature of lung metastasis, being a possible explanation of this phenomenon [35,[38][39][40].The signature genes identified include the EGFR ligand epiregulin, COX2 and matrix metalloproteinases 1 and 2 [41].Expression of these genes is important for motility but also allows angiogenesis, and intravasation of tumor cells into the circulation, which may be a factor of lung extravasation via the lung capillaries [40,42].Epiregulin and EGFR are of particular interest with several studies having found a strong correlation with their expression in TNBC [43][44][45][46][47][48].The expression of EGFR has been reported as being inversely related to hormone receptor expression [43,49,50], suggesting that lung metastasis may be an intrinsic feature of TNBC.
Our results showed that the DRFS was 30% and 10% at three and five years, respectively.Due to the lack of specific targets, treatment with standard agents leaves patients prone to systemic and local relapse [48].Cho et al. proposed that high expression of EGFR as well mutations of BRCA1 are linked to a worse RFS in TNBC [51].Further evidence for EGFR as a marker for unfavourable prognosis is that expression of the aforementioned gene is linked to resistance to chemotherapy and radiation treatment, inherently forewarning poor survival [49,50,52].
We found no significant difference in disease-free metastasis, regardless of the organ invaded.This might be explained by the overall inefficiency of the available treatments.Treatment options are limited to standard chemotherapy and there is a current lack of targeted therapies.Despite an apparent responsiveness to these agents, the prognosis remains poor due to the higher likelihood of visceral relapse; a phenomenon referred to as the TNBC paradox [53][54][55].This statement is demonstrated in the study by Fornier et al. [56]; who reported rapid progression following multiple lines of chemotherapy.
Regarding the stratification of risk factors according to the sites of metastasis, we found that a family history of breast or ovarian cancer was associated with higher rates of metastasis to multiple sites.Family history as a risk factor is mainly due to the association with BRCA1 mutations [57], which have been identified as contributors to the metastatic and aggressive nature of tumor cells [58].Fasching et al. [57] reported that brain metastasis was frequently seen in patients with BRCA1 mutations whereas Song et al. [59] found that carriers of this mutation frequently experience lung and lymph node metastasis.On the same hand, N1 tumor staging was associated with metastasis to visceral, CNS, and multiple sites.Similar to our results, Lin et al. described that lower lymph node involvement (N1) was associated with a greater risk of metastasis to solid organs such as the brain and lung [60].Finally, the association between death and metastasis to CNS and multiple sites showed the greatest significance.As shown in Table 3, multiple metastasis was related to death in 97.4% of cases whereas CNS and visceral metastasis to were associated with death in 88.5% and 85.6% of cases, respectively.
The average OS in the present study was lower than that described in a previous study (26% at 5 years) [61].It has been reported, that the outcomes of Latin American breast cancer patients are worse compared to patients from developed countries.These poor outcomes are mainly associated with socioeconomic factors that not only limit access to screening, favoring late diagnosis, but also cause deficient access to specialised cancer care [15,16].This should be one of the major priorities for intervention in our setting to achieve better short and long term outcomes in our population.
When comparing the different sites of metastases, the lowest OS rates were found in patients with multiple metastases, followed by CNS, visceral, and bone.Supporting the latter, Wang et al. and other authors found that patients with metastasis to visceral, CNS and multiple sites had a lower OS, whilst those with bone metastasis had the highest OS amongst [61][62][63].This last finding, together with the fact that TNBC tends to invade visceral sites more frequently than bone [64], is a framework to establish that worse prognosis and unfavorable OS in metastatic TNBC, are related to the excess risk and high incidence of visceral metastasis [27].Therefore, the invasion of multiple viscera burdens carries the worst prognosis due to life threatening and life diminishing clinical presentations [63,65].
We found that metastasis to CNS, N staging and multiple metastasis were prognostic factors for OS in TNBC.It was expected that a well-documented prognostic factor such as lymph node status [66][67][68], would be significant in multivariate analysis.In a study conducted using the SEER database, Gao et al. found that TNBC patients with distant metastasis have worse OS if they were IV stage, uninsured, did not undergo surgery and chemotherapy, and had metastasis in brain and liver [69].However, to our knowledge this is the first instance in which multiple metastasis has been identified as such a significant prognostic factor for OS and MS in metastatic TNBC.

Limitations
This was a retrospective study and thus, the associations among the factors analysed can be assured but not causation.Some patients were lost during follow-up, and were thereby excluded from the study, limiting maximum data collection and analysis.Moreover, test results for BRCA gene mutation was not available in the majority of our patients due to noncoverage by national insurance.In addition, although we included neoadjuvant and adjuvant chemotherapy, and radiotherapy in our analyses, we did not have further therapeutic information such as the chemotherapy regimen This information should be included in future studies due to the possible effects on the outcomes of this specific population.
Although the data used belonged to the main Peruvian Cancer Institute, patients from private centres might have different outcomes.Therefore, future investigations should include the collection of data from multiple centres in order to limit potential selection bias and achieve true randomization as well as being more applicable to other populations.

Conclusions
In conclusion, patients with multiple sites of metastasis have a 2.5-fold higher risk of death compared to those with bone metastasis in terms of OS and MS.Moreover, CNS metastasis is also a predictor of a worse OS compared to bone metastasis.There was no difference in DRFS regarding the different solid organs or according to type of metastasis.Further prospective randomized controlled studies are needed in TNBC patients presenting metastasis to assess different therapeutic modalities and molecular biomarkers.

Fig 3 .Fig 4 .
Fig 3. Comparison of overall survival of patients with triple negative breast cancer with distant relapse (A) and metastasis survival since the diagnosis of first metastasis (B) according to solid organ metastasis.https://doi.org/10.1371/journal.pone.0293833.g003 and Stata 17 (College Station, TX: StataCorp LLC).

Table 3 . Sociodemographic, clinical, pathological, surgical characteristics and outcomes of patients with triple negative breast cancer with distant relapse according to the type of metastasis. Characteristics Type of metastasis Total N = 390 p value Visceral N = 160 Central Nervous System N = 78 Bone N = 38 Multiple sites N = 114
b p-value calculated with chi-square https://doi.org/10.1371/journal.pone.0293833.t003

Table 4 . Cox regression analysis for the overall survival of patients with triple-negative breast cancer with distant metastasis with tumor stage I to III.
https://doi.org/10.1371/journal.pone.0293833.t004

Table 5 . Cox regression analysis of the survival of patients with triple-negative breast cancer with distant metastasis with tumor stage I to III following the diagno- sis of metastasis. Characteristics Survival since the diagnosis of the relapse
FHBOC: Family history of breast and/or ovarian cancer; HR: hazard ratio; CI: confidence interval.https://doi.org/10.1371/journal.pone.0293833.t005